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GFP Imaging Illuminating Science, One Protein at a Time by Daniel Haldar

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GFP Imaging Illuminating Science, One Protein at a Time by Daniel Haldar Focus: Illuminating Science Figure 1. Structure of green fluorescent protein. The second- ary structure of the protein is shown by beta sheets, while the chromophore is depicted by the small molecular spheres in the protein’s center. GFP Imaging Illuminating Science, One Protein at a Time By Daniel Haldar ioluminescence has intrigued field of biomedical research with scientists for years: how did the Bgenerations of human ob- its relevant and far-ranging appli- desiccated remains of Cypridina (a servers over the centuries: ancient cations. With GFP as an imaging mollusk) fluoresce when exposed Eastern civilizations wrote about tool, scientists are now capable of to water? (1). In essence, Hirata the firefly, the ancient Greeks re- visualizing important processes that wanted Shimomura to isolate and ported on light-producing marine were previously nearly impossible characterize the luciferin found in organisms, and even Shakespeare difficult to trace in fields ranging Cypridina. Even by present-day referenced the “effectual fire of from the study of cancer metastasis technological standards, Shimomura the glow-worm” in Hamlet. It is, to nerve cell damage. faced a daunting challenge because however, not until the 20th Cen- this project involved the purification tury that scientists have harnessed Shimomura: a Pioneer of of the luciferin out of the several the power of bioluminescence to Fluorescence Imaging thousands of compounds within make important scientific advances. GFP’s discovery began in post- the organism. Hirata’s assignment Indeed, Osamu Shimomura, Martin World War II Japan where Osamu of such a difficult project to a new Chalfie, and Roger Y. Tsien were Shimomura was working under the lab assistant seems counter-intuitive; jointly awarded the 2008 Nobel guidance of Nagoya University’s however, since Shimomura was Prize in Chemistry for their dis- Professor Yashimasa Hirata. As merely a lab assistant and not a covery and characterization of the a visiting assistant in Hirata’s lab, graduate student constrained by green fluorescent protein (GFP), a Shimomura worked on a project time limitations, Hirata felt that bioluminescent tool derived from that attempted to answer a puzzling there was nothing to lose with his Credit: http://commons.wikimedia.org/wiki/File:Gfp_and_fluorophore.png) jellyfish that has revolutionized the question whose answer had eluded decision (1). 20 Harvard Science Review • spring 2010 20-23.indd 20 2/17/2010 2:56:54 AM Focus: Illuminating Science After a year of remarkable work, Shimomura and Johnson pub- Shimomura defied expectations lished a paper in 1962, which de- by successfully crystallizing the lineated their methodology behind Cypridina luceferin and characteriz- purifying aequorin and described ing some of its chemical properties. their experimental observations Impressed by Shimomura’s accom- about aequorin’s chemical proper- plishments, Dr. Frank Johnson of ties. In addition, they also reported Princeton University’s Department that the purification of aequorin of Biology offered him a position yielded another protein that fluo- in his lab to study the properties resced green when exposed to UV of another bioluminescent organ- light (2). This finding became ism, Aequorea victoria (jellyfish). the first piece of evidence that Before Shimomura departed for proved the existence of what is the USA to accept Johnson’s po- now known as green fluorescent sition, Hirata requested Nagoya protein, or GFP. In his later work, University to grant Shimomura a Shimomura conducted an in- Figure 2. Expression of GFP in C. elegans. In postdoctoral degree although he depth examination of the chemical this particular study led by Guy Caldwell at was not technically in a graduate the University of Alabama, GFP was used in mechanisms that govern GFP’s a C. elegans system to study the effects of degree program (1). fluorescent capabilities. His work a protein called torsinA on the development of the neurological disorder torsion dystonia. In the summer of 1961, Shimo- revealed that GFP’s fluorescence mura and Johnson traveled from stems from the unique nature of cell development in the model or- Princeton to the Friday Harbor its chromophore, a chemical group ganism, C. elegans (roundworm). Laboratories in Washington to that selectively absorbs light and Chalfie intended to link the gene obtain the necessary specimens gives color to molecules (3). As that encoded GFP to a variety of of Aequorea to conduct their ex- UV light strikes the chromophore promoters (sections of DNA that periments. During the course of the direct transcription of genes) within summer, Shimomura and Johnson of GFP, it absorbs light to reach By Daniel Haldar an excited energetic state; when the C. elegans genome. Through collected thousands of jellyfish and this linkage, Chalfie hoped to map excised their light organs (1). When this energy is released, photons of light at the green wavelength are nerve cell activity by observing the they filtered these samples through location of promoters activated dur- rayon gauze, a transiently lumi- emitted. Unlike other fluorescent ing cellular processes (4) nescent fluid termed “squeezate” proteins such as aqueorin, the GFP The first step in Chalfie’s ex- was acquired. Through accidental chromophore is especially remark- perimental plan was to identify the circumstances, Shimomura made able because its functionality is location of the gene that encoded a breakthrough discovery when he not dependent on the presence of for GFP in the DNA of Aequorea. observed that the squeezate emitted other energy-donating chemical an intense light signal after being compounds (3). Douglas Prasher, a molecular bi- exposed to seawater because the ologist at the Woods Hole Oceano- calcium ions present in the aqueous Chalfie: Surprising graphic Institution, had already solution served as activators for a Observations conducted preliminary experiments fluorescent chemical mechanism. Martin Chalfie’s groundbreaking to locate and clone the GFP gene. After processing 10,000 specimens work with GFP began after he at- In 1988, Chalfie contacted Prasher of jellyfish, Shimomura and John- tended a seminar by Paul Brehm about a prospective collaboration son returned to Princeton to con- in 1988 on bioluminescence (4). and exchanged thoughts about duct further experiments on these Inspired by the signaling capa- various approaches that they could light-emitting processes. Within a bilities afforded by GFP, Chalfie employ to clone the GFP coding few months, they successfully puri- decided to integrate the protein’s gene (4). In 1989, however, Chalfie fied around 5 mg of protein, which functionality into his existing work went on sabbatical and lost contact they named aequorin, from the at Columbia University. At this with Prasher for a few years. Even- squeezate (1). time, Chalfie was studying nerve tutally, Chalfie stumbled upon on http://www.ninds.nih.gov/news_and_events/news_articles/news_article_torsion_dystonia.htm5. Credit: spring 2010 • Harvard Science Review 21 20-23.indd 21 2/17/2010 2:56:54 AM Focus: Illuminating Science one of Prasher’s publications and Tsien: the Color Spectrum cally. This observation allowed discovered that Prasher had finished Expands Tsien to conclude that GFP chro- identifying GFP’s genomic sequence Roger Tsien’s achievement of the mophore formation was depen- (5). After Chalfie rekindled his col- Nobel Prize is a fitting capstone for dent on the presence of oxygen laboration with Prasher to obtain a a research career whose foundations as an auxiliary factor (8). sample of the sequence, a graduate were built on visualizing cellular Tsien continued his work with student in Chalfie’s lab, Ghia Eu- processes right from the start. As GFP by introducing mutations skirchen, was able to express the a graduate student at the University at various points in the protein’s GFP gene in E.coli within a month’s of Cambridge, Tsien worked on a genomic sequence. These muta- time. When exposed to UV light, project in which he studied and syn- tions created significant changes these E.coli cells displayed green flu- thesized fluorescent labels for vari- in GFP’s emission spectrum: while orescence, indicating that the gene ous cellular messaging compounds, the primary peak of the light waves had been successfully expressed such as cyclic AMP and calcium GFP emits is normally located in (4). Other scientists had attempted ions (7). Because these molecules the UV region of the spectrum, similar approaches with Prasher’s are involved in a wide gamut of Tsien was able to make it appear sequence, but they were not suc- intracellular interactions – from in the visible blue region. By cessful in achieving these results. muscle movement to hormonal sig- continuing to make amino acid For instance, other scientists had naling – tagging such molecules with mutations, Tsien created a range mistakenly used inappropriate con- fluorescent labels allows scientists to of GFP mutants with varying structs to amplify Prasher’s DNA trace the mechanisms of dynamic spectral characteristics and fluo- sequence. In Chalfie’s laboratory, cellular processes. rescent colors (7). Having fluo- however, copies of only the coding Tsien soon realized that the chem- rescent markers of various colors region of the gene were formed us- ical labels he created during his is extremely useful for scientists ing PCR amplification (a molecular graduate studies lacked universal ap- because it facilitates the concurrent biology technique used to replicate plicability. For every new molecule tracking of proteins involved in specific stretches of DNA) to avoid he wished to study, Tsien had to different processes within the cell. interference from the contiguous synthesize a new type of fluorescent Furthermore, by using different sequences of non-coding DNA (4). label. To overcome the inefficiency fluorescent proteins, scientists can Although Chalfie and Euskirch- of this method, Tsien searched for en’s observation established GFP’s a protein that could function as an imaging tool, the serve as a universal phenomenon was rather surprising marker of gene expres- at the time of the discovery.
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